VLK drives extracellular phosphorylation of EphB2 to govern the EphB2-NMDAR interaction and injury-induced pain.

Phosphorylation of hundreds of protein extracellular domains is mediated by two kinase families, yet the significance of these kinases is underexplored. Here, we find that the presynaptic release of the tyrosine directed-ectokinase, Vertebrate Lonesome Kinase (VLK/Pkdcc), is necessary and sufficient for the direct extracellular interaction between EphB2 and GluN1 at synapses, for phosphorylation of the ectodomain of EphB2, and for injury-induced pain. Pkdcc is an essential gene in the nervous system, and VLK is found in synaptic vesicles, and is released from neurons in a SNARE-dependent fashion. VLK is expressed by nociceptive sensory neurons where presynaptic sensory neuron-specific knockout renders mice impervious to post-surgical pain, without changing proprioception. VLK defines an extracellular mechanism that regulates protein-protein interaction and non-opioid-dependent pain in response to injury.

Activation of α 6 -containing GABA A receptors induces antinociception under physiological and pathological conditions.

ABSTRACT: The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α 6 -containing GABA A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α 6 -containing GABA A receptor blockade or transient α 6 -containing GABA A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α 6 subunit is expressed in IB4 + and CGRP + primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α 6 subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α 6 -containing GABA A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α 6 subunit reduces tactile allodynia and restores α 6 subunit expression in neuropathic rats. Positive allosteric modulators of the α 6 -containing GABA A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α 6 subunit is expressed in humans. This receptor is found in CGRP + and P2X3 + primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α 6 -containing GABA A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.

Transient receptor potential canonical 5 mediates inflammatory mechanical and spontaneous pain in mice.

Tactile and spontaneous pains are poorly managed symptoms of inflammatory and neuropathic injury. Here, we found that transient receptor potential canonical 5 (TRPC5) is a chief contributor to both of these sensations in multiple rodent pain models. Use of TRPC5 knockout mice and inhibitors revealed that TRPC5 selectively contributes to the mechanical hypersensitivity associated with CFA injection, skin incision, chemotherapy induced peripheral neuropathy, sickle cell disease, and migraine, all of which were characterized by elevated concentrations of lysophosphatidylcholine (LPC). Accordingly, exogenous application of LPC induced TRPC5-dependent behavioral mechanical allodynia, neuronal mechanical hypersensitivity, and spontaneous pain in naïve mice. Lastly, we found that 75% of human sensory neurons express TRPC5, the activity of which is directly modulated by LPC. On the basis of these results, TRPC5 inhibitors might effectively treat spontaneous and tactile pain in conditions characterized by elevated LPC.

Convergence of peptidergic and non-peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn.

Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long-standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1, and Nav1.7 proteins stained the entirety of lamina 1-2, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences.

Pharmacological Manipulation of Translation as a Therapeutic Target for Chronic Pain.

Dysfunction in regulation of mRNA translation is an increasingly recognized characteristic of many diseases and disorders, including cancer, diabetes, autoimmunity, neurodegeneration, and chronic pain. Approximately 50 million adults in the United States experience chronic pain. This economic burden is greater than annual costs associated with heart disease, cancer, and diabetes combined. Treatment options for chronic pain are inadequately efficacious and riddled with adverse side effects. There is thus an urgent unmet need for novel approaches to treating chronic pain. Sensitization of neurons along the nociceptive pathway causes chronic pain states driving symptoms that include spontaneous pain and mechanical and thermal hypersensitivity. More than a decade of preclinical research demonstrates that translational mechanisms regulate the changes in gene expression that are required for ongoing sensitization of nociceptive sensory neurons. This review will describe how key translation regulation signaling pathways, including the integrated stress response, mammalian target of rapamycin, AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase-interacting kinases, impact the translation of different subsets of mRNAs. We then place these mechanisms of translation regulation in the context of chronic pain states, evaluate currently available therapies, and examine the potential for developing novel drugs. Considering the large body of evidence now published in this area, we propose that pharmacologically manipulating specific aspects of the translational machinery may reverse key neuronal phenotypic changes causing different chronic pain conditions. Therapeutics targeting these pathways could eventually be first-line drugs used to treat chronic pain disorders. SIGNIFICANCE STATEMENT: Translational mechanisms regulating protein synthesis underlie phenotypic changes in the sensory nervous system that drive chronic pain states. This review highlights regulatory mechanisms that control translation initiation and how to exploit them in treating persistent pain conditions. We explore the role of mammalian/mechanistic target of rapamycin and mitogen-activated protein kinase-interacting kinase inhibitors and AMPK activators in alleviating pain hypersensitivity. Modulation of eukaryotic initiation factor 2α phosphorylation is also discussed as a potential therapy. Targeting specific translation regulation mechanisms may reverse changes in neuronal hyperexcitability associated with painful conditions.

Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photoinactivate targeted proteins of interest. First, we show that protease-activated receptor 2 (PAR2), a G-protein-coupled receptor and an important pathway that leads to pain sensitization, can be photoinactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched off in laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Second, we demonstrate that the photoinactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood-brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification.

Limbic system development underlies the emergence of classical fear conditioning during the third and fourth weeks of life in the rat.

Classical fear conditioning creates an association between an aversive stimulus and a neutral stimulus. Although the requisite neural circuitry is well understood in mature organisms, the development of these circuits is less well studied. The current experiments examine the ontogeny of fear conditioning and relate it to neuronal activation assessed through immediate early gene (IEG) expression in the amygdala, hippocampus, perirhinal cortex, and hypothalamus of periweanling rats. Rat pups were fear conditioned, or not, during the third or fourth weeks of life. Neuronal activation was assessed by quantifying expression of FBJ osteosarcoma oncogene (FOS) using immunohistochemistry (IHC) in Experiment 1. Fos and early growth response gene-1 (EGR1) expression was assessed using qRT-PCR in Experiment 2. Behavioral data confirm that both auditory and contextual fear continue to emerge between PD 17 and 24. The IEG expression data are highly consistent with these behavioral results. IHC results demonstrate significantly more FOS protein expression in the basal amygdala of fear-conditioned PD 23 subjects compared to control subjects, but no significant difference at PD 17. qRT-PCR results suggest specific activation of the amygdala only in older subjects during auditory fear expression. A similar effect of age and conditioning status was also observed in the perirhinal cortex during both contextual and auditory fear expression. Overall, the development of fear conditioning occurring between the third and fourth weeks of life appears to be at least partly attributable to changes in activation of the amygdala and perirhinal cortex during fear conditioning or expression. (PsycINFO Database Record